DNA construct for enhancing the efficiency of transcription

ABSTRACT

Novel transcription initiation regions that provide for enhanced transcription of a DNA sequence, particularly a plant sequence, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 395,155,filed Aug. 17, 1989, now U.S. Pat. No. 5,164,316, which is acontinuation of U.S. application Ser. No. 147,887, filed Jan. 25, 1988,now abandoned, which is a continuation-in-part of application Ser. No.002,780, filed Jan. 13, 1987 now abandoned.

TECHNICAL FIELD

The present invention relates to DNA constructs that permit variation inenhancement of transcription initiation rate, particularly in plants.

BACKGROUND OF THE INVENTION

The ability to manipulate gene expression would provide a means ofproducing new characteristics in transformed plants. There are manysituations for which high or increased levels of gene expression may berequired; for example the production of important proteins foragronomical or commercial purposes, or the regulation of endogenous geneexpression by competing levels of anti-sense RNA. The enhancement oftranscription from promoters would facilitate these possibilities.Promoters vary in their transcription initiation rate and/or efficiency.Besides the transcription levels from a promoter other factors such asregulated or enhanced transcription should be considered. It istherefore of interest to provide DNA constructs which provideflexibility in the levels of expression obtained and under theappropriate conditions in which they are required to be effective.

DESCRIPTION OF THE RELEVANT LITERATURE

Barton et al., Cell (1983) 32:1033-1043, Caplan et al., Science (1983)222:815-821, Nester et al., Ann. Rev. Plant Physiol. (1984) 35:387-413,and Fraley et al., Biotechnology (1985) 3:629-635 describe the genetictransformation of dicotyledonous plants by the transfer of DNA fromAgrobacterium tumefaciens (A. tumefaciens) through the mediation ofmodified Ti plasmids. Odell et al., Nature (1985) 313:810-812 describethat the cauliflower mosaic virus (CaMV) 35S promoter is constitutivelyactive in several different species of plants. Bevan et al., EMBO J.(1985) 4:1921-1926 and Morelli et al., Nature (1985) 315:200-204describe that the CaMV 35S promoter is transcribed at a relatively highrate as evidenced by a ten-fold increase in transcription products incomparison to the NOS promoter. Abel et al., Science (1986) 232:738-743,Bevan et al., EMBO J. (1985) 4:1921-1926, Morelli et al., Nature (1985)315:200-204, and Shah et al., Science (1986) 233:478-481 describe thatthe 35S promoter is moderately strong and constitutively active, and sohas been used to express a number of foreign genes in transgenic plants.Odell et al., supra, describe that correct initiation of transcriptionfrom the 35S promoter is dependent on proximal sequences which include aTATA element, while the rate of transcription is determined by sequencesthat are dispersed over 300 bp of upstream DNA. Simpson et al., Nature(1986) 323:551-554 describe an enhancer region. (Sequences whichactivate transcription are termed enhancers.)

SUMMARY OF THE INVENTION

The present invention provides a novel transcription initiation regioncomprising an enhancer domain and, under the enhancing control of theenhancer domain, a transcription initiation domain. The enhancer domaincomprises a plurality of the repetitive units of a natural enhancerspaced in substantially the same way as a natural enhancer. In theabsence of a heterologous transcription initiation domain, the enhancerdomain has at least one more repetitive unit than the natural enhancer.The transcription initiation domain or promoter comprises an RNApolymerase binding site and an mRNA initiation site. The transcriptioninitiation regions of the present invention provide for an enhancedtranscription efficiency as compared to the promoter in the absence ofthe enhancer domain.

DNA constructs are also provided comprising a transcription initiationregion and a DNA sequence, which may be the RNA coded sequences of agene or RNA sequences of opposite orientation in relation to thewild-type transcription initiation domain (anti-sense sequence),particularly a plant sequence. The constructs provide for an enhancedefficiency of transcription of the RNA coded regions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates diagrammatically the organization of genes inintermediate vectors pCKR1 and pCKR2 and transgenic plants.Abbreviations used in the Figure are NPTII, neomycin phosphotransferase,type II B, BamHI; EV, EcoRV; H, HindIII; P, PstI; (EV/P), fusion ofEcoRV and PstI sites;

FIG. 2 is a diagrammatic structure of pCDX-1.

FIG. 3A is a diagrammatic structure of pCE7;

FIG. 3B is a diagrammatic structure of pCD7;

FIG. 4A is a diagrammatic structure of pCa1G7; and

FIG. 4B is a diagrammatic structure of pCa2G7;

FIG. 5 is a diagrammatic structure of pNC1 with the inserts for pNC2 andpNC4;

Abbreviations used in FIGS. 2-5 include, for restriction sites El EcoRl; H, HindIII-Hind III; P, Pst I; X, XbaI; B, BamHI; C, ClaI; S, SmaI;Xh, XhoI; K, KpnI; Sp, SphI; Ssp, SspI; N, NheI; A, AluI; EV, EcoRV;

For other sequences: G7--gene 7; Po--promoter; Sp^(r) --streptomycinresistance; N PTII--neomycin phosphotransferase II; Ca2promoter--Cauliflower mosaic virus 35S promoter with upstream enhancerduplication; nos--nopaline synthase.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides a novel transcription initiation region,particularly chimaeric or heterologous region, comprising an enhancerdomain and a transcription initiation domain under the enhancing controlof the enhancer domain. The transcription initiation regions of thepresent invention are useful in enhancing the transcription efficiencywhich may result in enhanced transcription of DNA sequences undercontrol of the transcription initiation region. Of particular interestis enhanced transcription of inserted gene sequences which may be of thesame genetic origin as the host or of foreign origin, either thenaturally occurring sequences (in both sense and antisense orientations)or synthetically prepared sequences.

The subject enhancer domains comprise a plurality of the repetitiveunits of a natural enhancer spaced in substantially the same way as thenatural enhancer, including the natural or wild-type enhancer. Theenhancer domain comprises at least the same number of the repetitiveunits as the natural enhancer and need be no more than the number ofrepeated elements for the optimal expression required, usually not morethan about three times the number of the repetitive units of the naturalenhancer. When the transcription initiation region does not include aheterologous transcription initiation domain (promoter), the enhancerdomain has at least one more repetitive unit than the natural enhancer.A natural enhancer comprises a DNA sequence which in its nativeenvironment is upstream from and within about 600 bp of a promoter.Taking the initial nucleotide of the mRNA as 0, the sequence containingan enhancer is from about -50 to about -1000 bp, usually from about -50to -950 bp, generally comprising about -100 to -800 bp.

The subject DNA enhancer sequences contain repetitive units comprisingshort sequences of from about 4 to about 16 bp, including four unit,seven unit and ten unit repeats, which are repeated within the sequencecomprising the enhancer. The repeat will usually include at least theunit GTGG (CACC), although in some instances one of the G's may bereplaced by an A, particularly the second G. Exemplary of suchrepetitive units is the sequence GTGG (or complementary sequence CCAC),##STR1## the former two being repeated several times within the upstreamsequence of the CaMV 35S promoter region. The repeats may be imperfect,having a specific core and some degree of variability in the surroundingsequence. The repeats may be used in combination or as independentrepeat sequences, that is the same or different sequences may berepeated.

By similarly spaced to a natural enhancer, it is meant that the numberof nucleotides between the repetitive units is substantially the same asin the natural enhancer. This will generally be at least about 4 and notmore than 100 nt. Usually the repetitive units will be separated by notmore than the largest number of nucleotides separating the repetitiveunits in the natural enhancer and not fewer than the smallest number ofnucleotides separating the repetitive units in the natural enhancer.More usually, the repetitive units in the enhancer domain will have anaverage spacing which is within about 16 bp, more usually within about 4bp, of the average spacing of the repetitive units in the naturalenhancer. Conveniently, the spacers between repetitive units may be thesame as the wild-type enhancer, recreating the wild-type enhancer andextending the wild-type enhancer with a fragment of or a completeenhancer region. The spacer nucleotides may be the same or differentfrom the wild-type spacer nucleotides and may be in the same ordifferent order from the wild-type spacer order.

For the most part the enhancer domain will have at least about 4,usually at least about 6, and preferably at least about 8 repeats of the4 bp sequence, and a total of at least about 3, usually at least about4, of the 7 and 10 bp sequences. Generally, there will be from about 4to 100 bp separating the repeat sequences. Of course, since GTGG ispresent in the longer sequences, in referring to the number of repeatsequences, the requirements for GTGG should not be read into therequirements for the longer sequences.

The enhancer domain will usually be at least about 25 bp, more usuallyat least about 30 bp, generally at least about 50 bp and not more thanabout 1 kbp, usually not more than about 700 bp, frequently not morethan about 600 bp.

The enhancer sequence will come from sources which function efficientlyin eukaryotes, particularly of higher multicellular orders ofeukaryotes, more particularly plants and animals, usually plants.Usually the enhancer will be of viral or (higher) eukaryotic origin. Theenhancer may be used in the same or different kingdom, genus or speciesfrom which it is derived or in which it naturally functions.

Many viral, plant and animal enhancers contain sets of repeated sequenceelements (repetitive units) (Shaffner, Eukaryotic Transcription: theRole of cis-and trans-acting Elements in Initiation (1985) ed. Y.Gluzman, 1-18). For example, several short (4 to 7 bp) sequences arerepeated within the upstream sequences of the CaMV 35S promoter. Amongthese, the sequences GTGG (and its complement CCAC) and ##STR2## aredistinguished by being selectively localized adjacent to and upstreamfrom the two promoter regions of the CaMV genome. The 270 bp of DNAupstream from the TATA element of the 35S promoter contains ten copiesof these sequences and the 19S promoter has four copies dispersed over150 bp of DNA immediately upstream from its TATA element. The remaining7600 bp in the CaMV genome contains only 26 GTGG sequences (Franck etal., Cell (1980) 21:285-294).

GTGG sequences are concentrated upstream from or within the promoterregions of many plant genes (Coruzzi et al., EMBO J. (1984) 3:1671-1679,Dean et al., EMBO J. (1985) 4:3055-3061, Doyle et al., J. Biol. Chem.(1986) 261:9228-9238, Fluhr et al., Science (1986) 232:1106-1112,Herrera-Estrella et al., Nature (1984) 310:115-120, and Sommer andSaedler, Mol. Gen. Genet. (1986) 202:429-434). A subset of thesesequences have attracted attention (Odell et al. (1985) supra, Coruzziet al. (1984) supra, Dean et al. (19850 supra, Doyle et al. (1986)supra, Fluhr et al. (1986) supra, and Kaulen et al., EMBO J. (1986)5:1-) because they resemble the sequence ##STR3## which is common tomany of the enhancers associated with viral and animal genes (Weiher etal., Science (1983) 219:626-631). Extensive upstream sequences of somelight-regulated plant promoters which contain GTGG sequences canactivate transcription when positioned in either orientation upstream ofa heterologous promoter Fluhr et al. (1986), supra, and Timko et al.,Nature (1985) 318:579-582. However, such an upstream promoter regionexcised from a ribulose bisphosphate carboxylase small subunit gene hadno significant enhancing effect when placed downstream of a heterologouspromoter (Timko et al. (1985) supra). Similarly an upstream element ofthe light harvesting protein gene (lhcp) gene gave no enhancedexpression when placed 3' to the expressed sequences (Simpson et al.Nature (1986) 323:551-554).

An enhancer domain is cis-acting and desirably is located within about5000 bp, usually about 2000 bp, more usually adjacent to or within about1000 bp of a transcription initiation domain to be enhanced. Theenhancer may be in either orientation, e.g., ##STR4## with respect tothe transcription initiation domain and can be located upstream ordownstream in relation to the promoter it enhances, usually upstream. (ais 0 or 1, and b is 0 or 1.)

An enhancer domain of the present invention finds use with a widevariety of initiation domains, including promoters that are naturallyfound under the control of the enhancer, i.e., in a cis position(adjacent and homologous) and those not normally associated with theparticular promotor (i.e., heterologous).

The enhancer domain and transcription initiation domain may be from thesame or different kingdom, family or species or a pathogen whichfunctions in the cell source of the initiation region (promoter).Species of interest include prokaryotes and eukaryotes, such asbacteria, plants, insects, mammals, etc. Combinations may includeenhancer domains from a virus with a transcription inititiation regionof a structural gene of a host for the virus; an enhancer domain fromone species with a gene (structural gene and promoter) from a differentspecies of the same or different family; etc.

The invention also contemplates DNA constructs comprising a subjecttranscription initiation region and, under the control of thetranscription initiation region, a DNA sequence to be transcribed. TheDNA sequence may comprise a natural open reading frame includingtranscribed 5' and 3' flanking sequences. Alternatively, it may comprisean anti-sense sequence in that it encodes the complement of an RNAmolecule or portion thereof. When the construct includes an open readingframe (ORF) which encodes a protein, an enhanced transcriptioninitiation rate is obtained, usually providing an increased amount ofthe polypeptide expression product of the gene. When the constructcomprises an anti-sense sequence, the enhanced transcription of RNAcomplementary to wild type suppresses the expression of the wild typemRNA, thereby decreasing the amount of the polypeptide expressionproduct. In addition, antisense RNA can also function as an inhibitor ofreplication of RNA (of viral genomes for example).

Enhanced transciption in plants may find use in enhancing the productionof proteins characteristic of the plant (endogenous) or those proteinsfrom other genetic sources (exogenous). (By endogenous is intendednormally found in the wild-type host, while exogenous intends notnormally found in the wild-type host.)

The initiation regions of the subject invention may be used in a varietyof contexts and in combination with a variety of sequences, "RNA codedsequences." The RNA coded sequences of a gene may be those of a naturalgene, including the open reading frame for protein coding and frequentlythe 5' and 3' untranslated sequences. The RNA translational initiationsequences are included in the constructs, either from the promoterdomain or from the attached coding sequences.

The RNA coded sequence will include:

1. Protein encoding sequences of a gene (plant, animal, bacterial,viral, and fungal) which may be employed include: (a) natural openreading frame encoding a protein product; (b) complementary DNA (cDNA)sequences derived from mRNA of a gene; (c) synthetic DNA giving theappropriate coding sequence; (d) protein encoding sequences derived fromexons of the natural gene, i.e., open reading frame produced by exonligation; (e) combinations thereof.

Attached to the above sequences are appropriate transcriptiontermination/polyadenylation sequences.

2. Sequences from the natural gene (plant, animal, bacterial, viral, andfungal) which encode the primary RNA product, i.e., consisting of exonsand introns, e.g., natural Polymerase II and Polymerase III transcribedgenes of eukaryotes.

3. Synthetic DNA sequences which encode a specific RNA or proteinproduct.

4. Sequences of DNA modified from known (natural gene) coding sequencesby mutagenesis, for example, site specific mutagenesis.

5. Chimeras of any of the above achieved by ligation of DNA fragments,e.g., which encode fused proteins.

6. DNA sequences encoding the complement of RNA molecules or portionsthereof.

Examples of the above sequences to be expressed from thepromoter-enhancer constructs of the subject invention include: antisenseRNA (for gene suppression); nutritionally important proteins; growthpromoting factors; proteins giving protection to the plant under certainenvironmental conditions, e.g., proteins giving resistance to metal orother toxicity; stress related proteins giving tolerance to extremes oftemperature, freezing, etc.; compounds of medical importance, e.g.,anti-microbial, anti-tumor, etc.; proteins of specific commercial value;increased level of proteins, e.g., enzymes of metabolic pathways,increased levels of products of structural value to a plant host, e.g.,herbicide resistance, such as increased amounts of EPSP synthase (Shahet al., Science (1986) 233:417), pesticide resistance, e.g., B.thuringiensis toxin, etc. The sequences of interest which aretranscribed will be of at least about 8 bp, usually at least about 12bp, more usually at least about 20 bp, and may be one or more kbp.

In some instances it will be desirable to delete the untranslatedupstream region of the gene at a site fewer than 250 bp (-250) from theinitiation codon (+1), usually fewer than 150 bp, more usually fewerthan 100 bp, but not less than about 50 bp, more usually not less thanabout 70 bp. Truncation will be of particular interest where an invertedrepeat of a total of at least 8 bp, usually at least 10 bp is present inthe region. Usually, the truncation will be upstream from the CAAT boxof the upstream promoter region.

Conveniently the DNA construct can additionally include a selectionmarker. Exemplary of markers useful in plant cells are bleomycin,hygromycin, kanamycin resistance, e.g. neomycin phosphotransferase typeII gene (which permits selective growth of transformed plant tissue inthe presence of kanamycin or G418), and glucuronidase. Exemplary of suchmarkers useful in animal cells are thymidine kinase, dihydrofolatereductase, those identified above, etc.

The subject constructs will be prepared employing cloning vectors, wherethe sequences may be naturally occurring, mutated sequences, syntheticsequences, or combinations thereof. The cloning vectors are well knownand comprise prokaryotic replication systems, markers for selection oftransformed host cells, and unique dual restriction sites for insertionor substitution of sequences. For transcription and optimal expression,base DNA may be transformed into a host, e.g. mammalian host, forintegration into the genome, where the subject construct is joined to amarker for selection or is co-transformed with DNA encoding a marker forselection. Alternatively, episomal elements (plasmids) may be employedwhich may be introduced into the host cell by transformation,transfection, transduction, conjugation, electroporation, fusion, etc.where the episomal element may be stably maintained at a copy number of1 or greater or may be integrated into the host cell genome.

The manner in which the subject constructs are introduced into the hostcell is not critical to this invention. Nor is it critical that theconstruct be an extrachromosomal element or integrated into the hostgenome. With plants the construct may be introduced using A. tumefaciensusing a T-DNA construct with left and right borders, electroporation,microinjection, etc. With mammalian cells, transformation ortransfection may be employed.

In a particular embodiment of the invention, the enhancer domaincomprises a modified CaMV 35S enhancer with a heterologous promoter or35S promoter or an unmodified 35S enhancer with a heterologous promoter.Desirably, an enhancer domain is employed having at least one morerepetitive unit than the natural CaMV 35S enhancer, desirably having two35S enhancer domains in tandem in either orientation. Illustrative ofheterologous promoters are Ti-plasmid promoters, such as the T-DNA gene5 and 7 promoters.

The constructs may be used with cells in suspension culture, tissue,organisms, e.g. plants, callus, or the like.

A variant of the CaMV 35S enhancer promoter in which the sequencesupstream of the TATA element were tandemly duplicated was constructed.Transcription from the 35S promoter in transgenic plants was increasedten-fold by this modification. The duplicated upstream region of theCaMV 35S promoter also acted as a strong transcriptional enhancer ofheterologous promoters, increasing the transcription rate of an adjacentand divergently transcribed T-DNA gene, i.e., gene 7, severalhundredfold and, to a lesser extent, that of another T-DNA gene from aremote downstream position. This engineered enhancer element increasedthe expression of foreign genes in transgenic plant cells and plants.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

The efficiency of transcription of the natural CaMV 35S promoter wasraised by duplicating the transcription-activating sequences upstream ofthe TATA element, as shown in FIG. 1. The natural and duplicated 35Spromoters were transferred to tobacco plants for functional analysis bythe use of intermediate vectors derived from pMON178. Preparation ofthose vectors is described in detail, below. Briefly, pMON178 is amodification of pMON129 (Fraley et al., Proc. Natl. Acad. Sci. USA(1983) 80:4803-4807) containing a nopaline synthase (NOS) gene, achimaeric neomycin phosphotransferase type II (NPT) gene which permitsselective growth of transformed plant tissue in the presence ofkanamycin, and an 1800 bp fragment from the transferable region (T-DNA)of an octopine-type Ti plasmid (FIG. 1). The NPT-encoding sequences ofpMON178 are linked to a promoter that was derived from the NOS gene.This promoter was excised and replaced by 35S promoters with single ordouble copies of the upstream region to make the intermediate vectorspCKR1 or pCKR2 (FIG. 1).

The CaMV 35S sequences are from the CAbb BS strain, as reported byFranck et al., Cell (1980) 21:285-294.

Structures of Genes in Intermediate Vectors and in Transgenic Plants.

Vectors were prepared as illustrated in FIG. 1. Abbreviations used inthe Figure are B, BamHI; EV, EcoRV; H, HindIII; P, PstI; (EV/P), fusionof EcoRV and PstI sites. A fragment of the 35S promoter extendingbetween positions -343 to +9 was previously constructed in pUC13 byOdell et al., supra. It was excised as a ClaI-HindIII fragment, madeblunt-ended with DNA polymerase I (Klenow fragment), and inserted intothe HincII site of pUC18 to make pCA1. The upstream region of the 35Spromoter was excised from this plasmid as a HindIII-EcoRV fragment andinserted into pCA1 between the HindIII and PstI (blunt) sites to makepCA2. The unrearranged or duplicated 35S promoters were excised frompCA1 or pCA2, respectively, as HindIII-BamHI fragments and insertedbetween the HindIII and BgIII sites of pMON178 to make the intermediatevectors pCKR1 or pCKR2.

Intermediate vectors were transferred from E. coli to A. tumefaciens bytriparental mating. Those that had integrated into resident Ti plasmidsby recombination were selected as described (Rogers et al., Methods inEnzymol. (1986) 118:627-640). The recombination event resulted inlinkage of the intermediate vector sequences to Ti plasmid sequencesincluding the left T-DNA border and a complete promoter for gene 5. (SeeWillmitzer et al., EMBO J. (1982) 1:139-146). After infection of leafdiscs, the DNA between the right (RB) and the left (LB) border sequencesof the recombinant Ti plasmid was transferred to and chromosomallyintegrated into the plant cells, with the transferred genes arranged asshown at the bottom of FIG. 1. The open boxes represent promoters andarrows represent transcribed regions.

The intermediate vectors were established in A. tumefaciens byhomologous recombination with resident copies of the disarmedoctopine-type Ti plasmid pTiB6S3SE as described previously (Rogers etal. (1986) supra). The recombinant plasmids contained the octopine-typeT-DNA genes 5 and 7 (Willmitzer et al. (1982) supra) (structural detailsof T-DNA gene 7, including the complete DNA sequence are described inMcPherson, Nuc. Acids Res. (1984) 12:2317-2325) and the NPT and NOSgenes of the intermediate vector, all of which are flanked by right andleft T-DNA border sequences which delineated the region of the Tiplasmid that was integrated into the genome of transformed plant cells.

Leaf discs of Nicotiana tabacum cv. xanthi H38 were infected withcultures of recombinant A. tumefaciens and cultured in vitro asdescribed (Rogers et al. (1986) supra). Tobacco cells carrying stablyintegrated copies of the NPT gene and associated DNA were selected bygrowth of calli and subsequent shoot development in the presence of 300μg/ml of kanamycin. Individual shootlets were excised from thetransformed tissue and grown to a height of 40 to 60 cm. Analysis of DNAisolated from leaves showed that one to five intact copies of each ofthe four transferred genes were present in the genomes of theregenerated plants.

NPT and T-DNA Gene 7 Transcripts in Transgenic Plants

(Throughout the text gene 7 and gene 5 refer to the T-DNA genes. SeeMcPherson, Nucleic Acids Res. (1984) 12:2317-2325.)

The amounts and initiation sites of transcripts of the transferred geneswere determined by their protection of single-stranded DNA probes fromdigestion by S1 nuclease.

To determine the amount of transcription, leaves were ground in a mortarin 25 mM EDTA pH 7.5, 75 mM NaCl, 1% SDS, and extracted twice withphenol and once with isobutanol. Liberated nucleic acids wereprecipitated by the addition of 0.1 volume of 3 M sodium acetate pH 5.5and 2 volumes of ethanol. RNA was recovered from the nucleic acidsamples by precipitation with LiCl as described (McPherson et al., Proc.Natl. Acad. Sci. USA (1980) 77:2666-2670). Single-stranded, continuouslylabelled DNA probes were synthesized, hybridized with 2 μg of leaf RNA,digested with S1 nuclease, and analysed by electrophoresis as described(Kay et al. (1986) Mol. Cell Biol. 6: 3134-31.

The 35S/NPT probe was synthesized from an M13mp18 template containing aninsert of the HindIII-PvuII fragment of the 35S/NPT gene isolated frompCKR1, and was excised from template DNA by digestion with EcoRV. Thegene 7 probe was synthesized from an M13mp18 template containing theHindIII-PvuII fragment of gene 7 isolated from the HindIII Y fragment ofthe octopine Ti plasmid pTiB6 806 (Gelvin et al., Proc. Natl. Acad. Sci.USA (1982) 79:76-80), and was excised by digestion with HindIII.

Transcripts in total leaf RNA were identified and quantified by S1nuclease protection. The four plants of each transformed type are arepresentative selection of the six to ten plants of each type that wereexamined for gene expression. Equivalent levels of transcriptionalactivation by the unrearranged and duplicated 35S promoters wereobserved in additional sets of plants derived from independenttransformation experiments. When linked to a NOS promoter (pMON178), theNPT-encoding sequences were transcribed at low levels in the leaves oftransgenic plants.

Replacement of the NOS promoter by a wild-type CaMV 35S promoter (CKR1)resulted in a ten-fold increase on average in the number of NPTtranscripts. However, the modified 35 S promoter with a duplicatedupstream region (CKR2) had by far the highest activity, producingroughly one hundred times as many NPT transcripts as did the NOSpromoter.

The promoter of the chimaeric NPT gene was positioned immediatelyupstream of the divergently transcribed gene 7 in the transgenic plants(FIG. 1). With a NOS promoter linked to the NPT-encoding sequences, gene7 was transcribed at barely detectable levels, as described above. Gene7 transcription was increased about forty-fold above these levels by thepresence of an upstream and divergently oriented 35 S enhancer andpromoter. A further ten-fold increase in gene 7 transcription wasinduced when the upstream region of the NPT-linked 35S enhancer regionwas duplicated. In this situation the enhancer sequence (the upstreamregion of the 35S promoter) served as a bidirectionalpromoter-activating element.

The proportions of NPT and gene 7 transcripts were not the same amongdifferent plants that had been transformed with the same DNA, even whenthese two genes were being simultaneously activated by the 35S upstreamregion (enhancer sequence). Variability in rates of transcription amongeither closely linked or widely separated genes within the same fragmentof transferred DNA is commonly observed in transgenic plants (Jones etal., EMBO J. (1985) 4:2411-2418, Nagy et al., EMBO J. (1985)4:3063-3068, and Velten et al., Nucleic Acids Res. (1985) 13:6981-6998);the causes of such variation in gene expression are unknown.

In addition to activating transcription of the proximal 35S and gene 7promoter elements, the duplicated region upstream of the 35S promoterwas able to greatly increase transcription at a pair of initiation sitesin the gene 5 promoter, despite being located about 2000 bp downstreamof it, as described below.

Gene 5 Transcripts in Transgenic Plants

Transcripts in 2 μg of leaf RNA were identified and quantified by S1nuclease protection of probes as described previously. Two classes ofGene 5 transcripts were identified, initiating about 320 bases (+1) and300 bases (+20) upstream of the RsaI site. The probe was synthesizedfrom an M13mp18 template containing an insert of the HindIII-RsaIfragment of gene 5 isolated from the HindIII Y fragment of the octopineTi plasmid pTiB6 806 (Gelvin et al., Proc. Natl. Acad. Sci. USA (1982)79:76-80), and was excised at the SstI site in the M13mp18 polylinkerbeyond the insert.

The remote activation of the gene 5 promoter identifies the upstreamregion of the 35S promoter as a true transcriptional enhancer (Khoury etal., Cell (1983) 33:313-314, and Shaffner, Eukaryotic Transcription: theRole of cis-and trans-acting Elements in Initiation (1985) ed. Y.Gluzman, 1-18).

The transcript levels of gene 5 were much lower than those of gene 7when both were being activated by the duplicated 35S enhancer. Thisreflects the natural difference in the strengths of their promoters(Willmitzer et al. (1982) supra and Gelvin et al. (1982) supra) but thegreater distance between the enhancer and the gene 5 promoter could alsoresult in a lesser degree of transcriptional activation, as observedwith other enhancers (Wasylyk et al., Nucleic Acid Res. (1984)12:5589-5608).

Transcription of the NOS gene was not significantly affected by thepresence of a duplicated 35S enhancer at a remote and downstreamlocation on the same fragment of transferred DNA, as described below.

NOS Gene Transcripts in Transgenic Plants

Transcripts in 2 μg of leaf RNA were identified and quantified by S1nuclease protection of probes as described previously. The probe wassynthesized from an M13mp19 template containing an insert of theEcoRI-HpaII fragment of the NOS gene isolated from pMON178, and wasexcised by digestion with NdeI.

Construction of Other Vectors

The following exemplary DNA constructs comprising transcriptioninitiation regions of the present invention were prepared.

A modified version of pMON316 (Rogers et al., Biotechnology in PlantSciences: Relevance to Agriculture in the Nineteen Eighties (1986)Academic Press 219), called pCDX1, which has duplicated 35S enhancersequences upstream of a polylinker insertion site and a polyadenylationsignal sequence was constructed as illustrated in FIG. 2.

Construction of pCDX1

The duplicated 35S enhancer domain was excised from pCA2 as aHindIII-BamH1 fragment and ligated to the BglIII-BstEII fragment of pMON316 (containing the T-DNA right border) and the BstEII-Eco RI fragmentof pMON 200 (containing the Nos-NPT gene). The EcoRI and HindIII siteswere made blunt ended with DNA polymerase 1 (Klenow fragment) andligated together to give the final plasmid, pCDX1.

Promoter-enhancer Constructs Combining the Enhancer Sequences of CaMV35S and the Promoter of T-DNA Gene 7.

A. Construction of regions CE7 and CD7 in plasmids pCE7 and pCD7.

The MnlI (270 bp) fragment containing upstream sequences and thepromoter of T-DNA gene 7 was excised from T-DNA cloned sequences. TheMnlI fragment was inserted into the SmaI site of pUC 18 to give pC7.

The HindIII fragment of pC7 containing the promoter region of T-DNA gene7 and adjacent polylinker was excised and inserted:

1. into the HindIII site upstream of the CaMV 35S enhancer region inpCa1 to align the T-DNA gene 7 promoter with the adjacent natural CaMV35S enhancer domain producing a "region", CE7 of plasmid pCE7 (FIG. 3A).

2. into the HindIII site upstream of the duplicated CaMV 35S sequencesin pCa2 to align the T-DNA gene 7 promoter with the adjacent duplicatedCaMV 35S enhancer domain producing a "region" CD7 of plasmid pCD7 (FIG.3B).

The enhancer-promoter regions CE7 and CD7 are also marked in FIGS. 4Aand 4B.

B. Plasmids pCa1G7 and pCa2G7

These plasmids facilitate the attachment of the regions CE7 and CD7 togene coding sequences (including termination sequences) via polylinkers.

pCa1G7 (FIG. 4A) and pCa2G7 (FIG. 4B) are constructed by initiallymaking a co-integrate plasmid by ligating together the followingfragments:

the BstEII-EcoRI fragment of pMON 200;

the EcoRI-AccI fragment of pC7;

and the ClaI-BstEII fragment of pMON 316.

The HindIII fragment of this plasmid, carrying the gene 7 promoter(i.e., HindIII-MnlI) and adjacent polylinker is excised and ligatedinto:

1. the HindIII site of pCa1 (to give pCa1G7);

2. the HindIII site of pCa2 (to give pCa2G7).

These intermediate vectors are useful for obtaining efficient expressionof protein-encoding sequences in transgenic plants. The vector pCKR2 canbe used for the same purpose if a complete transcription unit isinserted at the HindIII site of the vector with its promoter adjacent tothe 35S enhancer elements.

The extent of activation of a promoter by an enhancer appears to differfor different promoters.

Of the three heterologous promoters that were examined, the NOS promoterwas not as highly activated by the 35S enhancer. Therefore, promoterdomains may be screened for their responsiveness to a particularenhancer.

In the following examples, pNC1 was constructed by replacing theEcoRI-HindIII NOS/NPTII fragment of pMON178 with the EcoRI-HindIIINOS/NPT fragment of pNNSΔNC. pNNSΔNC was obtained from pNNS whichresulted from the insertion of the HpaI-HindIII fragment of pMON129 intothe SmaI-HindIII sites of pUC18. By deleting the upstream region -73 to-256 of the NOS promoter by digestion with NheI and ClaI and filling inwith the Klenow fragment followed by ligation, pNNSΔNC was obtained.

pNC2, -3 and -4 were constructed by inserting the CaMV HindIII fragmentof the pAV-H series (H1A, H1B and H2, respectively) into the HindIIIsite of pNC1. pAV-H plasmids have AluI to EcoRV sequences of the CaMV35S enhancer inserted into the HincII sites of pUC1813 (Kay andMcPherson, Nucleic Acids Res. (1987) 15:2778. pAV-H1A and -H1B have asingle insert in the (+) or (-) orientation, while pAV-H2 has a doubleinsert with both AluI-EcoRV copies in the same orientation. (See FIG.5.)

Alternative transcription initiation regions for enhanced expressionfrom the T-DNA gene 7 promoter and from the NOS initiation region (i.e.truncated promoter to -73) are represented in the constructs pNC2 andpNC4. The single upstream region of the CaMV 35S enhancer comprises 8 ofthe 10 GTGG or (CCAC) repeats previously associated with the CaMV 35Supstream region. Is also includes the three extended repeats ##STR5##i.e. at sites -100, -140, and -260. The duplicate of this region isrepresented in pNC4.

Levels of enhanced transcription from the T-DNA gene 7 promoter arehigher for the single construct than in CKR1. For expression levels ofgene 7, the duplicated form of the truncated 35S upstream region isequivalent to the duplicated form in CKR2.

The expression level from the truncated NOS promoter in the absence ofthe 35S upstream sequence (i.e. pNC1) was undetectable. In the presenceof a single (pNC2 or 3) or double (pNC4) copy of the EcoRV-AluI regionof the CaMV 35S upstream region, the enhancement of expression from theNOS initiation site was greater than that obtained with an intact NOSpromoter region, i.e. -258 to +1. These results support the conclusionthat sequences between -73 and -258 of the NOS upstream region have aninhibitory or regulatory effect on the CaMV 35S enhancer.

In accordance with the subject invention, novel DNA constructs areprovided which allow for increased rate of transcription initiation ofvarying degrees. Thus, employing the same promoter region, variouslevels of transcription initiation are achieved in the absence of anenhancer or the presence of a wild-type enhancer or extended enhancer.These constructions can have wide application in the control of theformation of expression products, protection against pathogens orantibiotics, etc.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed:
 1. A DNA construct having as components, (a) atranscription initiation region including (i) a tandemly duplicated CaMV35S enhancer sequence comprising an AluI-EcoRV fragment of a CaMV 35Supstream region; (ii) a promoter comprising an RNA polymerase bindingsite and an mRNA initiation site; (b) a nucleotide sequence of interestfor transcription to mRNA; and (c) a termination region wherein saidcomponents are operably joined.
 2. The DNA construct according to claim1, wherein said promoter is a T-DNA gene 7 or gene 5 promoter or a CaMV35S promoter.
 3. The DNA construct according to claim 1, furthercomprising as component (d) the right T-DNA border.
 4. The DNA constructaccording to claim 1, wherein said sequence of interest is an openreading frame with an initiation codon for expressing a polypeptide ofinterest.
 5. The DNA construct according to claim 1, wherein saidpromoter is a CaMV 35S promoter.